Reconstitution of β2‐adrenoceptor−GTP‐binding‐protein interaction in Sf9 cells

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Abstract
In most studies, coupling of the β2-adrenoceptor (β2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which β2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the β2AR alone, in combination with the α-subunit of Gs (Gsα), and as fusion protein with Gsα (β2AR-Gsα) in Sf9 insect cells. The β2AR expressed alone couples poorly to the endogenous Gsα-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5′-O-(3-thiotriphosphate) (GTP[S]) binding were small. β2AR-Gsα reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the β2AR co-expressed with a large excess of Gsα. In membranes expressing β2AR-Gsα, highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing β2AR and Gsα as separate proteins was difficult to detect. Our data show that the β2AR interacts with Gsα more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused Gsα. The β2AR-Gsα fusion protein provides a very sensitive model system to study the regulation of Gs function by β2AR agonists and inverse agonists directly at the level of the G-protein.

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